Gerard C. Blobe

Role of Transforming Growth Factor Beta in Human Cancer

Transforming growth factor beta (TGF-beta) is a ubiquitous and essential regulator of cellular and physiologic processes including proliferation, differentiation, migration, cell survival, angiogenesis, and immunosurveillance. Alterations in the TGF-beta signaling pathway, including mutation or deletion of members of the signaling pathway and resistance to TGF-beta-mediated inhibition of proliferation are frequently observed in human cancers. Although these alterations define a tumor suppressor role for the TGF-beta pathway in human cancer, TGF-beta also mediates tumor-promoting effects, either through differential effects on tumor and stromal cells or through a fundamental alteration in the TGF-beta responsiveness of the tumor cells themselves. TGF-beta and members of the TGF-beta signaling pathway are being evaluated as prognostic or predictive markers for cancer patients. Ongoing advances in understanding the TGF-beta signaling pathway will enable targeting of this pathway for the chemoprevention and treatment of human cancers.

Role of transforming growth factor-β superfamily signaling pathways in human disease

Transforming growth factor beta (TGF-beta) superfamily signaling pathways are ubiquitous and essential regulators of cellular processes including proliferation, differentiation, migration, and survival, as well as physiological processes, including embryonic development, angiogenesis, and wound healing. Alterations in these pathways, including either germ-line or somatic mutations or alterations in the expression of members of these signaling pathways often result in human disease. Appropriate regulation of these pathways is required at all levels, particularly at the ligand level, with either a deficiency or an excess of specific TGF-beta superfamily ligands resulting in human disease. TGF-beta superfamily ligands and members of these TGF-beta superfamily signaling pathways also have emerging roles as diagnostic, prognostic or predictive markers for human disease. Ongoing studies will enable targeting of TGF-beta superfamily signaling pathways for the chemoprevention and treatment of human disease.

Mechanical Stiffness Grades Metastatic Potential in Patient Tumor Cells and in Cancer Cell Lines

Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here, we applied a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacologic inhibition of myosin II increases invasiveness, whereas increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power-law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.

Regulation of protein kinase C and role in cancer biology

Protein kinase C (PKC) is a family of closely related lipid-dependent and diacyglycerol-activated isoenzymes known to play an important role in the signal transduction pathways involved in hormone release, mitogenesis and tumor promotion. Reversible activation of PKC by the second messengers diacylglycerol and calcium is an established model for the short term regulation of PKC in the immediate events of signal transduction. PKC can also be modulated long term by changes in the levels of activators or inhibitors for a prolonged period or by changes in the levels of functional PKC isoenzymes in the cell during development or in response to hormones and/or differentiation factors. Indeed, studies have indicated that the sustained activation or inhibition of PKC activityin vivo may play a critical role in regulation of long term cellular events such as proliferation, differentiation and tumorigenesis. In addition, these regulatory events are important in colon cancer, where a decrease in PKC activators and activity suggests PKC acts as an anti-oncogene, in breast cancer, where an increase in PKC activity suggests an oncogenic role for PKC, and in multidrug resistance (MDR) and metastasis where an increase in PKC activity correlates with increased resistance and metastatic potential. These studies highlight the importance and significance of regulation of PKC activityin vivo.

Arachidonic acid and free fatty acids as second messengers and the role of protein kinase C

In addition to serving as the precursor to a plethora of eicosanoids and other bioactive molecules, arachidonate may function as a bona fide second messenger. A number of studies have documented the ability of arachidonate to regulate the function of multiple targets in vitro systems. This has been particularly well established and studied with the activation of protein kinase C by arachidonate in a mechanism distinct from activation by diacylglycerol. In cells, arachidonate induces a number of activities, many of which may be independent of further metabolism to eicosanoids; suggesting possible direct action of arachidonate. This review summarizes the current state of knowledge on the possible second messenger function of arachidonate with specific emphasis on the regulation of protein kinase C.

The type III TGF-β receptor suppresses breast cancer progression

TheTGF-β�signalingpathwayhasacomplexroleinregulatingmammarycarcinogenesis.�Herewedemon- stratethatthetypeIIITGF-β�receptor�(TβRIII,�orbetaglycan),�aubiquitouslyexpressedTGF-β�coreceptor,� regulatedbreastcancerprogressionandmetastasis.�MosthumanbreastcancerslostTβRIIIexpression,�with� lossofheterozygosityoftheTGFBR3�genelocuscorrelatingwithdecreasedTβRIIIexpression.�TβRIIIexpres- siondecreasedduringbreastcancerprogression,�andlowTβRIIIlevelspredicteddecreasedrecurrence-free� survivalinbreastcancerpatients.�RestoringTβRIIIexpressioninbreastcancercellsdramaticallyinhibited� tumorinvasivenessinvitroandtumorinvasion,�angiogenesis,�andmetastasisinvivo.�TβRIIIappearedto� inhibittumorinvasionbyundergoingectodomainsheddingandproducingsolubleTβRIII,�whichbinds� andsequestersTGF-β�todecreaseTGF-β�signalingandreducebreastcancercellinvasionandtumor-induced� angiogenesis.�OurresultsindicatethatlossofTβRIIIthroughallelicimbalanceisafrequentgeneticevent� duringhumanbreastcancerdevelopmentthatincreasesmetastaticpotential.

Bone Morphogenetic Proteins Signal through the Transforming Growth Factor-β Type III Receptor

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-β (TGF-β) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-β type III receptor (TβRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-β and inhibin receptor. Here we demonstrate that TβRIII functions as a BMP cell surface receptor. TβRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-β. TβRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TβRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TβRIII function, we demonstrate that BMP-2 specifically stimulates TβRIII-mediated epithelial to mesenchymal cell transformation. The ability of TβRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-β signaling suggests a broader role for TβRIII in orchestrating TGF-β superfamily signaling.

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

The Type III Transforming Growth Factor-β Receptor as a Novel Tumor Suppressor Gene in Prostate Cancer

The transforming growth factor-beta (TGF-beta) signaling pathway has an important role in regulating normal prostate epithelium, inhibiting proliferation, differentiation, and both androgen deprivation-induced and androgen-independent apoptosis. During prostate cancer formation, most prostate cancer cells become resistant to these homeostatic effects of TGF-beta. Although the loss of expression of either the type I (TbetaRI) or type II (TbetaRII) TGF-beta receptor has been documented in approximately 30% of prostate cancers, most prostate cancers become TGF-beta resistant without mutation or deletion of TbetaRI, TbetaRII, or Smads2, 3, and 4, and thus, the mechanism of resistance remains to be defined. Here, we show that type III TGF-beta receptor (TbetaRIII or betaglycan) expression is decreased or lost in the majority of human prostate cancers as compared with benign prostate tissue at both the mRNA and protein level. Loss of TbetaRIII expression correlates with advancing tumor stage and a higher probability of prostate-specific antigen (PSA) recurrence, suggesting a role in prostate cancer progression. The loss of TbetaRIII expression is mediated by the loss of heterozygosity at the TGFBR3 genomic locus and epigenetic regulation of the TbetaRIII promoter. Functionally, restoring TbetaRIII expression in prostate cancer cells potently decreases cell motility and cell invasion through Matrigel in vitro and prostate tumorigenicity in vivo. Taken together, these studies define the loss of TbetaRIII expression as a common event in human prostate cancer and suggest that this loss is important for prostate cancer progression through effects on cell motility, invasiveness, and tumorigenicity.

Increased Toxicity With Gefitinib, Capecitabine, and Radiation Therapy in Pancreatic and Rectal Cancer: Phase I Trial Results

PURPOSE Overexpression of epidermal growth factor receptor (EGFR) has been associated with aggressive tumor phenotypes, chemotherapy, and radiation resistance, as well as poor survival in preclinical and clinical models. The EGFR inhibitor gefitinib potentiates chemotherapy and radiation tumor cytotoxicity in preclinical models, including pancreatic and colorectal cancer. We initiated two phase I trials assessing the combination of gefitinib, capecitabine, and radiation in patients with localized pancreatic and rectal cancer. PATIENTS AND METHODS Patients with pathologically confirmed adenocarcinoma of the pancreas and rectum were eligible. Pretreatment staging included computed tomography, endoscopic ultrasound, and surgical evaluation. Patients received 50.4 Gy of external-beam radiation therapy to the tumor in 28 fractions. Capecitabine and gefitinib were administered throughout the radiation course. Following completion, patients were restaged and considered for resection. Primary end points included determination of dose-limiting toxicity (DLT) and a phase II dose; secondary end points included determination of non-DLTs and preliminary radiographic and pathologic response rates. RESULTS Ten patients were entered in the pancreatic study and six in the rectal study. DLT was seen in six of 10 patients in the pancreatic study and two of six patients in the rectal study. The primary DLT in both studies was diarrhea. Two patients developed arterial thrombi. CONCLUSION The combination of gefitinib, capecitabine, and radiation in pancreatic and rectal cancer patients resulted in significant toxicity. A recommended phase II dose was not determined in either of our studies. Further investigation with this combination should be approached with caution.